Abstract Polymorphic antigens on RBCs contribute to the high incidence of allo-immunization and to the presence of multiple antibodies in the serum of sickle cell disease (SCD) patients. Identifying the specificity of these antibodies is critical for providing compatible blood. However, the current process can be complex, involve costly reference laboratory testing, lacks standardization, and is hampered by the lack of suitable reagent RBCs for timely provision of transfusion service. The major goals of the parent application to this revision application are to develop a panel of reagent cultured RBCs (cRBCs) specifically targeted for SCD patients to identify the presence of clinically significant antibodies to donor antigens. This panel of reagent cRBCs will be produced from iPSCs derived from donors carrying rare blood groups. We have made significant progress toward this goal and are now proposing to expand the scope of the parent application by accomplishing three novel Aims. In Aim 1, we propose to optimize cRBC production in a stirred mini-bioreactor setting which has become affordable because we have developed R5, an inexpensive, chemically-defined, albumin-free medium to differentiate erythroid progenitors into enucleated RBCs. In Aim 2, we will characterize in details and optimize the growth conditions of the 704D cell line, an iPSC-derived immortalized erythroid cell line that can differentiate into cRBCs that we recently developed by targeting dCAS9-VP64 to the promoter of an endogenous immortalizing gene to activate it in an inducible manner. The 704D cell line was generated by transducing iPSCs with lentiviral vectors leading to variable expression levels of the transduced genes. In order to generate a panel of immortalized erythroblasts that can be used to produce RBCs, we need to reproducibly engineer cell lines similar to 704D from each of the donors that constitute our proposed panel of cRBCs. We propose in Aim 3 to generate these cell lines using a site-specific integration approach that obviate the problems associated with lentiviruses. Accomplishments of these three Aims will accelerate the development of our panel of reagent cRBCs.